164 The Origin of Oocyte, In Vitro-Matured Oocyte With/Without Super-Stimulation, and In Vivo-Matured Oocyte Influence the Timing of Cleavage in Early Embryo and Oxygen Consumption of Blastocyst After IVF in Japanese Black Cow

Abstract

It has been reported that in vitro- and in vivo-matured oocyte obtained from fully growth follicles have high developmental competence. Furthermore, the timing of cleavage in early embryo after IVF affect pregnancy success after embryo transfer. It is still unknown whether origin of oocyte affects the timing of cleavage. In this study, we examined the influence of oocyte origin on cleavage timing of early embryo after IVF. Japanese Black cows were used as donors. Oocytes derived from non-stimulation follicles (control: CON), fully grown follicles after super-stimulation treatment (SST) and follicles just before ovulation after ovulation-induction treatment (in vivo-matured oocyte: VIVO) were obtained by ovum pick-up (OPU). In the CON group, OPU was conducted on arbitrary days except oestrus. In SST group, dominant follicles were aspirated and a CIDR was inserted into the vagina on Day 0, and then FSH was injected twice a day from the evening of Day 1 to the morning of Day 5 with decreasing doses in total 20 AU. In the evening of Day 4, prostaglandin F_2α (0.5 mg of cloprostenol) was administered. On Day 6, SST oocytes were collected after CIDR withdrawl. In the VIVO group, the treatment was carried out as SST until prostaglandin F_2α administration, and then CIDR withdrawal and administration of gonadotropin-releasing hormone (GnRH, 0.2 mg of fertirelin acetate) performed on the evening of Day 4 and morning of Day 5, respectively. The VIVO oocytes were collected at 25 to 26 h after GnRH. The CON and SST oocytes were inseminated after 20 to 22 h of IVM, and VIVO oocytes were inseminated at 30 h after GnRH, with 3 × 10⁶ sperm mL⁻¹, respectively. After 6 h of IVF, presumptive zygotes were individually cultured for 168 h, using a well-of-the-well dish (Dai-Nippon-Print, Japan) and were observed by time-lapse cinematography (CCM-4MZS; Astec, Japan) to analyse the cleavage timing of embryos. Oxygen consumption (O₂) was measured in blastocysts on 168 hpi with a scaning electrochemical microscopy system (HV-405SP; Hokuto Denko, Japan). Statistical analysis was carried out by Steel-Dwass test for the timing of cleavage and Tukey-Kramer test for O₂. In CON (n = 15), SST (n = 25), and VIVO (n = 36), the time of first cleavage was 27.5, 29.1, and 26.1 hpi, that of second cleavage was 38.9, 40.3, and 36.0 hpi, and that of third cleavage was 48.5, 46.1, and 45.9 hpi, respectively. These cleavage times were shorter in VIVO than in CON and SST (P < 0.01). The time interval between first and second cleavage (2nd cell cycle) was shorter in VIVO (10.1; P < 0.01) than CON (11.4) and SST (11.2). The time interval between second and third (3rd cell cycle) were shorter (P < 0.01) in SST (9.4) than in VIVO (10.1), and in VIVO than in CON (10.2), respectively. Consumption of O₂ was lower (P < 0.01) in CON (0.61 × 10⁻¹⁴ mol s⁻¹) than in SST (0.94 × 10⁻¹⁴ mol s⁻¹) and VIVO (0.94 × 10⁻¹⁴ mol s⁻¹). These results suggest that the origin of oocyte influences the length of cell cycle and O₂ consumption of blastocyst producted in vitro.